CD40 is a 48 kDa transmembrane glycoprotein cell surface receptor that shares sequence homology with the tumor necrosis factor alpha (TNFα) receptor family and was initially identified as a B-cell surface molecule that induced B-cell growth upon ligation with monoclonal antibodies (Durie, et al. (1994) Immunology Today 15:406-411; Vogel & Noelle (1998) Semin. Immunol. 10:435-442; Banchereau, et al. (1994) Ann. Rev. Immunol. 12:881-922). Dendritic cells, macrophages, epithelial cells, hematopoietic progenitors, and non-hematopoietic cells have been shown to express CD40. Its ligand, CD154, is a 34-39 kDa type II integral membrane protein expressed on activated but not resting T cells, activated B cells (Higuchi, et al. (2002) J. Immunol. 168:9-12), and activated platelets (Henn, et al. (1998) Nature 391:591-594; Danese, et al. (2003) Gastroenterology 124:1249-1264). During inflammatory responses, other cell types such as peripheral blood monocytes, human vascular endothelial cells, smooth muscle cells and mononuclear phagocytes (Bavendiek, et al. (2002) J. Biol. Chem. 277:25032-25039) have all been shown to express CD154.
Blocking CD154 ligand, and thus CD40/CD154 ligation, is an effective means by which to induce transplantation tolerance (Montgomery, et al. (2001) Immunol. Rev. 183:214-222; Yamada & Sayegh (2002) Transplantation 73:S36-39). Prevention of transplant rejection by blocking CD40/CD154 interactions has been repeatedly documented for the induction of long-term tolerance to skin (Quezada, et al. (2003) Blood 102:1920-1926; Elster, et al. (2001) Transplantation 72:1473-1478; Gordon, et al. (1998) Diabetes 47:1199-1206; Markees, et al. (1997) Transplantation 64:329-335; Jarvinen, et al. (2003) Transplantation 76:1375-1379; Quezada, et al. (2004) Annu. Rev. Immunol. 22:307-328), islets (Benda, et al. (2002) Cell Transplant. 11:715-720), bone marrow (Wekerle, et al. (2001) J. Immunol. 166:2311-2316), and a myriad of other transplanted organs (Camirand, et al. (2002) Transplantation 73:453-461; Tung, et al. (2003) Transplantation 75:644-650). These findings demonstrate the importance of this receptor-ligand pair in immunity and tolerance.
Peripheral tolerance is a constitutive process, whereby the immune system is constantly generating active suppressive immune responses to self in the periphery. Successful induction of peripheral tolerance relies on the quiescent state of self-antigen presenting antigen presenting cells, provision of self-antigens in the appropriate macromolecular form, and the emergence of regulatory T-cells to appropriate self-specificities. Through understanding these mechanisms, successful strategies have been developed to induce tolerance to alloantigens for the purpose of inducing transplant tolerance.
CD154 blockade alone and more commonly with other immunosuppressive interventions, can markedly delay graft rejection. It is widely held that one of the major effects of CD154 blockade is the interference with dendritic cell maturation. T-cell activation is inextricably linked to dendritic cell maturation based on the understanding of the signals that are necessary to induce T-cell expansion and differentiation. The two signal hypothesis of T-cell activation is an accepted paradigm that describes the early requirements for T-cell activation versus anergy (Mondino, et al. (1996) Proc. Natl. Acad. Sci. USA 93:2245-2252). This paradigm states that the proficient induction of an immune response requires T-cell receptor and MHC/peptide interaction (signal one) followed by the interaction between co-stimulatory molecules, namely CD80/86 and CD28 (signal two), and a possible myriad of other molecules. If signal one is generated in absence of signal two, the outcome is not immunity, but tolerance. Providing signal one and signal two becomes the responsibility of the antigen presenting cell, which must efficiently engage and trigger multiple T-cell surface molecules. To achieve this proficiency, antigen presenting cells must mature. Dendritic cell maturation by CD154 is unique in that it provides a spectrum of signals that trigger the upregulation of co-stimulatory molecules, cytokines, chemokines and allows for heightened dendritic life in vivo. Therefore, decision of tolerance versus immunity is centered, at least in part, on the maturational status of the antigen presenting cell compartment. Appropriate maturation of antigen presenting cells will license them to trigger productive cell-mediated immunity.
The impact of depriving dendritic cells of a maturational signal results in T-cell death, anergy and the emergence of regulatory T-cells (Treg) One way of exemplifying what the impact is of immature dendritic cell antigen presentation, is to examine the immune response to self-antigens. In a steady-state, dendritic cells from most lymphoid organs are phenotypically and functionally immature (Wilson, et al. (2004) Blood 103:2187-2195), whereas dendritic cells that migrate into the iliac, mesenteric, mediastinal, or subcutaneous lymph nodes from peripheral tissues are mature. Persistent presentation of self-antigens by immature dendritics is a critical factor in maintaining peripheral self-tolerance (Steinman, et al. (2003) Annu. Rev. Immunol. 21:685-711). This self-tolerance is induced by the induction of T-cell death, anergy and the emergence of self-reactive Treg. In particular, studies show that immature dendritic cells are capable of consuming cell-associated antigens and inducing T-cell anergy or deletion as long as they are not induced to mature via CD40 ligation (Bonifaz, et al. (2002) J. Exp. Med. 196:1627-1638; Hawiger, et al. (2001) J. Exp. Med. 194:769-779; Scheinecker, et al. (2002) J. Exp. Med. 196:1079-1090). It is believed that CD11c+, CD8αhi and not CD8αlo, are the dendritic cells that mediate peripheral tolerance. Other means of inducing Treg have also been cited as it has been recently found that in vivo targeting of antigen to immature dendritic cells via DEC205 leads to the expansion of CD4+CD25+CTLA4+ regulatory T-cells (Mahnke, et al. (2003) Trends Immunol. 24:646-651).
Much like the consumption of self-apoptotic cells, the infusion of allogeneic leukocytes, in the form of donor-specific transfusion (DST), in the presence of CD154 blockade facilitates the presentation of alloantigens by immature dendritic cells resulting in allospecific tolerance. It is well-known that form and biochemical makeup of the self-antigens (and possibly allogeneic antigens) is likely critical to the outcome of the events programmed by immature dendritic cells. It has been shown that the uptake of apoptotic cells by dendritic cells is an effective inducer of T-cell tolerance (Albert, et al. (1998) Nature 392:86-89; Inaba, et al. (1998) J. Exp. Med. 188:2163-2173), whereas the phagocytosis of necrotic cells has been proven to induce maturation of dendritic cells and development of immunity (Sauter, et al. (2000) J. Exp. Med. 191:423-434). The difference resides in the proinflammatory components released by necrotic cells (like heat shock proteins) which result in induction of the innate immune response (Vabulas, et al. (2002) J. Biol. Chem. 277:15107-15112; Vabulas, et al. (2002) J. Biol. Chem. 277:20847-20853), meanwhile clearance of apoptotic cells by specific cell surface receptors is accompanied by secretion of anti-inflammatory cytokines such as TGF-β (Huynh, et al. (2002) J. Clin. Invest. 109:41-50; Golpon, et al. (2004) FASEB J. 18 (14):1716-8). In addition, the capacity of necrotic or stressed apoptotic cells to induce dendritic cell maturation is evidenced by the enhanced expression of CD40 and costimulatory molecules, together with heightened IL-12 secretion (Kuppner, et al. (2001) Eur. J. Immunol. 31:1602-1609; Zeng, et al. (2003) Blood 101:4485-4491). Therefore, tolerance induction by DST inadvertently takes advantage of a pathway that is engineered to induce tolerance to self. DST in clinical practice utilizes whole blood to facilitate transplantation tolerance. While in virtually all mouse studies, spleen cells are used, it has been shown that the infusion of 200 μL of whole mouse blood three times over a period of one week, together with <CD154 induces profound graft tolerance.
Historically, the infusion of whole blood from the donor into graft recipients modestly prolonged allografts in humans (Flye, et al. (1995)Transplantation 60:1395-1401; Anderson, et al. (1995) Transplant Proc. 27:991-994) and mice (Wood, et al. (1984) J. Immunol. 132:651-655). The enhanced graft survival following DST can be substantially lengthened or rendered permanent if the DST is combined with blocking CD154, in both mice and monkeys, indicating the potential clinical relevance of this approach (Preston, et al. (2005) Am. J. Transplant. 5:1032-1041; Parker, et al. (1995) Proc. Natl. Acad. Sci. 92:9560-9564). Long-term survival of allogeneic kidneys in monkeys that are treated with αCD154, DST and rapamycin has been demonstrated (Kirk, et al. (1997) Proc. Natl. Acad. Sci. USA 94:8789-8794; Lin, et al. (1998) Mol. Cell. Biol. 18:5523-5532).
It is believed that infused DST rapidly undergoes apoptosis and is presented by host antigen presenting cells and αCD154 may facilitate the apoptosis of the DST by depriving it of a CD40 signal. At the same time, αCD154 impairs the maturation of host antigen presenting cells, committing them to the tolerogenic presentation of DST-derived allopeptides. Delivery of peptides via apoptotic cells appears to be an efficient means to induce peripheral tolerance. In addition, it has been shown that TAP−/− B-cells that are hyperosmotically-loaded with ovalbumin can induce abortive expansion and anergy of ovalbumin-specific cytotoxic T lymphocytes in vivo via indirect presentation (Liu, et al. (2002) Nat. Med. 8:185-189). Similarly, antigens expressed on dying pancreatic cells (Coulombe, et al. (1999) J. Immunol. 162: 2503-2510) induce tolerance via indirect presentation. Whether targeted via apoptotic cells, or by vectors that target directly to defined dendritic cell surface molecules (like DEC-205), antigens delivered to immature dendritic cells induce profound antigen-specific tolerance, which is amplified in the presence of αCD154.
αCD154-mediated tolerance via cytotoxic deletion of the activated, alloreactive T-cell population has been addressed. It has been shown in mice that (CD154 is ineffective in mice deficient in complement or the common FcR γ chain (Sanchez-Fueyo, et al. (2002) Transplantation 74:898-900; Monk, et al. (2003) Nat. Med. 9:1275-1280). It has also been shown that aglycosylated αhCD154 (reduced Fc and complement activation) is ineffective in managing graft rejection in primates, yet effective at blocking humoral immune responses (Ferrant, et al. (2004) Int. Immunol. 16:1583-1594). Moreover, at higher doses of Fab′2, αCD154 is quite effective at inducing alloreactive T-cell anergy and ablation. Furthermore, the impact of αCD154 on alloreactive T-cell contraction was completely reversed by co-administration of αCD40, suggesting that the T-cells were not eliminated. However, C′ and Fc-dependent mechanisms may be operative in this system, but it does not appear to be due to the direct deletion of alloreactive T-cells by αCD154.
Whether graft tolerance is induced by αCD154/DST, non-depleting αCD4 monoclonal antibodies, combinations of αCD154/CTLA-41g, or αCD2/αCD3 monoclonal antibodies, all induce Treg activities that are critical for long-term tolerance (Graca, et al. (2005) Trends Immunol. 26:130-135). Early studies described a “dominant” and “infectious” form of tolerance in a variety of allograft tolerance systems (Qin, et al. (1993) Science 259:974-977). Using DST and αCD154, it was shown that CD4+ T-cells were critical for long-lived survival of the allograft (Graca, et al. (2000) J. Immunol. 165:4783-4786). Subsequent studies in allogeneic bone marrow transplantation and other transplant models implicated an important role of CD4+CD25+ regulatory T-cells (Treg) in αCD154-induced graft tolerance (Hara, et al. (2001) J. Immunol. 166:3789-3796; Taylor & Namba (2001) Immunol. Cell Biol. 79:358-367; Taylor, et al. (2001) Blood 98:467-474; Taylor, et al. (2001) J. Exp. Med. 193:1311-1318).
Additional evidence substantiating the importance of Treg in long-lived graft tolerance have been provided by reconstitution studies. In these studies, RAG−/− mice were reconstituted with defined CD4+ T-cell populations, treated with DST and αCD154, and evaluated for whether graft tolerance could be induced following skin allograft. Upon reconstitution with only CD4+CD25−, T-cells, DST and αCD154 delayed significantly the rejection of allogeneic skin, but the grafts were ultimately rejected. In this case, the delay was due to extensive clonal abortion of the relevant alloreactive T-cells, however a small frequency remained that eventually rejected the graft. Upon the co-adoptive transfer of CD4+CD25+ T-cells with the CD25 T-cells, permanent graft survival becomes evident. Hence, clonal abortion of the alloreactive CD4+ effectors is incomplete, as there appears to be a CD4+CD25− population that is resistant to tolerance induction. The co-transfer of Treg can readily silence this residual population.
Natural CD4+CD25+ Treg, often referred to as naturally-occurring Treg, represents 5-10% of peripheral CD4+ T-cells in naïve mice and humans. It is believed that these cells emerge from the thymus as a consequence of high-affinity interactions with self-antigen, avoiding negative selection and escape to the periphery (Jordan, et al. (2001) Nat. Immunol. 2:301-306). The functional regulatory importance of this subset of cells was demonstrated by their critical role in maintaining peripheral tolerance (Takahashi & Sakaguchi (2003) Int. Rev. Cytol. 225:1-32; Itoh, et al. (1999) J. Immunol. 162:5317-5326; Takahashi, et al. (2000) J. Exp. Med. 192:303-310; Kumanogoh, et al. (2001) J. Immunol. 166:353-360; Shimizu, et al. (2002) Nat. Immunol. 3:135-142; Kanamaru, et al. (2004) J. Immunol. 172:7306-7314; Hori, et al. (2003) Adv. Immunol. 81:331-371; Takahashi & Sakaguchi (2003) Curr. Mol. Med. 3:693-706; Sakaguchi, et al. (2003) Novartis Found. Symp. 252:6-23, 106-114). Subsequent analysis in transplantation systems (Hara, et al. (2001) supra; van Maurik, et al. (2004) J. Immunol. 172:2163-2170; Graca, et al. (2000) supra; Taylor, et al. (2001) supra; Waldmann, et al. (2004) Semin. Immunol. 16:119-126; Lin, et al. (2002) Nat. Immunol. 3:1208-1213) and in autoimmunity and infectious disease (Mayer, et al. (1986) N. Engl. J. Med. 314:409-413) demonstrated the important regulatory function of these cells in vivo. Based on the surface phenotype of Treg (CD45RBlo, CTLA-4+, GITR+, CD62Lhi, CD25+), purification strategies have emerged. It appears, however, that the foxhead box P3 transcription factor (Foxp3) may be a specific molecular marker for this lineage (Brunkow, et al. (2001) Nat. Genet. 27:68-73; Hori, et al. (2003) Science 299:1057-1061; Fontenot, et al. (2003) Nat. Immunol. 4:330-336; Khattri, et al. (2003) Nat. Immunol. 4:337-342). Mice that report a fluorochrome when Foxp3 is transcriptionally activated have been produced (Fontenot, et al. (2005) Immunity 22:329-341; Wan & Flavell (2005) Proc. Natl. Acad. Sci. USA 102:5126-5131). Use of the Foxp3GFP/RFP mice (Fontenot, et al. (2005) supra) will facilitate the identification of Treg in vivo.
Functional assessment of Treg has relied extensively on the use of in vitro assays. A commonly used assay to measure Treg activity is the co-culture of Treg with Teff, with the measurement of suppression of Teff proliferation. This suppression has been shown to be due to a contact-dependent mechanism and not due to cytokines. It has been shown that the granzyme family of molecules may play an important role in contact-mediated suppression by Treg (Gondek, et al. (2005) J. Immunol. 174:1783-1786); Treg can directly kill Teff cells, and thus cell cytotoxicity may play a role in suppression.
In contrast to in vitro studies with Treg, in vivo studies have identified a number of important soluble factors in suppression. Recent studies have documented a role of IL-10 and TGFβ in tissue and solid graft tolerance (Quezada, et al. (2003) supra; Quezada, et al. (2004) supra; Graca, et al. (2000) supra; Waldmann, et al. (2004) supra; Kingsley, et al. (2002) J. Immunol. 168:1080-1086; Honey, et al. (1999) J. Immunol. 163:4805-4810). It has been in systems of co-stimulatory blockade (αCD154, CTLA-4-Ig, αCD4 (non-depleting)) that the role of Treg and these factors have been best illuminated. It has been shown that Treg can suppress the responses of both CD4+ and CD8+ effector T cells in graft rejection (van Maurik, et al. (2004) supra; Lin, et al. (2002) supra). Studies have shown that neutralizing IL-10 using αIL-10 or αIL-10R antibodies abrogate suppression and allow the rejection of skin (Hara, et al. (2001) supra; Kingsley, et al. (2002) supra), although there are studies at odds with this finding (Graca, et al. (2002) J. Immunol. 168:5558-5565). A role of IL-10 (but not IL-4) in the immunosuppression of inflammatory bowel disease by Treg has been demonstrated (Graca, et al. (2002) J. Immunol. 168:5558-5565). Further evidence of a need for IL-10 in colitis model, not a transplant model, was that Treg from IL-10−/− mice could not suppress colitis (Asseman, et al. (1999) J. Exp. Med. 190:995-1004).
TGFβ has also been implicated as an immunosuppressive mediator for Treg (Josien, et al. (1998) J. Clin. Invest. 102:1920-1926). In models of DST-induced tolerance in rats, high levels of TGFβ have been noted (Josien, et al. (1998) supra). In rat cardiac transplant models, neutralization of IL-10 and TGFβ has been shown to alleviate tolerance (Bickerstaff, et al. (2000) Transplantation 69:1517-1520). It has also been reported that Treg express surface TGFβ (Nakamura, et al. (2001) J. Exp. Med. 194:629-644; Kitani, et al. (2003) J. Exp. Med. 198:1179-1188), but later studies using Treg from TGFβ−/− mice or from mice with a dominant-negative TGFβ receptor have questioned the importance of Treg production of TGFβ in suppression (Pennica, et al. (1992) Biochem. 31:1134-1141). It has been reported that Treg may induce TGFβ from the host, and in that way mediate graft tolerance. Greater insights into the role of TGFβ have been provided in the Irritable Bowel Disease models where it has been reported that Treg from TGFβ−/− mice suppress colitis, and that Treg likely induce TGFβ liberation and activation from the host (Fahlen, et al. (2005) J. Exp. Med. 201:737-746).
In contrast to the naturally-occurring Treg, adaptive regulatory T cells (TR) have also been described. TR arise in the periphery from CD4+CD25− T-cells given particular cytokine environments (notably TGFβ), by presentation of immature dendritic cells or via particular routes of antigen administration (e.g., nasal routes (Chen, et al. (1994) Science 265:1237-1240)). As with Treg, TR also must be defined operationally. For practical purposes TR can be produced by the culture of CD4+CD25− T cells in vitro with TGFβ (Walker, et al. (2003) J. Exp. Med. 198:249-258; Zheng, et al. (2004) J. Immunol. 172:5213-5221; Zheng, et al. (2004) J. Immunol. 172:1531-1539; Horwitz, et al. (2003) J. Leukoc. Biol. 74:471-478; Zheng, et al. (2002) J. Immunol. 169:4183-4189; Chen, et al. (2003) Blood 101:5076-5083), by the over-expression of Foxp3 via retroviral transduction (Hori, et al. (2003) supra), or through selective means of antigen presentation in vitro (Jonuleit, et al. (2000) J. Exp. Med. 192:1213-1222; Sato, et al. (2003) Blood 101:3581-3589; Hoyne, et al. (2001) Immunol. Rev. 182:215-227) or in vivo (Apostolou, et al. (2002) Nat. Immunol. 3:756-763). It has also been reported in human systems that the co-culture of Treg with Teff can induce the Teff to become suppressive (Stassen, et al. (2004) Transplantation 77:S23-25).